title: Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.
abstract: Dynamic reprogramming of the genome takes place during the gamete-to-embryo transition. This transition defines a period of continuous and global change but has been difficult to study because of extremely limited material and varying degrees of chromatin compaction. Improved methods of detecting chromatin and gene expression changes in the germ line and in the preimplantation embryo would greatly enhance the understanding of this crucial developmental transition. Here we describe a protocol developed and used by us that improves the sensitivity of existing fluorescence in situ hybridization (FISH) methods; the protocol described here has enabled us to visualize single-copy DNA targets and corresponding nascent RNA transcripts in preimplantation embryos and during spermatogenesis. Major improvements over alternative methods involve fixation and permeabilization steps. Chromatin epitopes can be visualized simultaneously by combining FISH with immunofluorescence; multicopy and repetitive element expression can also be reliably measured. This procedure (sample preparation and staining) requires 1-1.5 d to complete and will facilitate detailed examination of spatial relationships between chromatin epitopes, DNA and RNA during the dynamic transition from gamete to embryo.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Howard Hughes Medical Institute, Boston, Massachusetts, USA. satoshi.namekawa@cchmc.org
authors.forename: Satoshi H
authors.initials: SH
authors.lastname: Namekawa
authors.forename: Jeannie T
authors.initials: JT
authors.lastname: Lee
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry; D002875:Chromosomes / Q000737:chemistry; D004247:DNA / Q000032:analysis*; D005854:Germ Cells / Q000737:chemistry*; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D051379:Mice; D012322:RNA Precursors / Q000032:analysis*; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity
chemical_list: D002843:Chromatin; D012322:RNA Precursors; D004247:DNA